A Molecular Epidemiological Study On HIV-1 Subtypes And Drug-resistance In Some Parts Of Jiangsu Province

Background and Objective: B/B',01_AE,07_BC and 08_BC subtypes are the main subtypes of HIV-1 in China nowadays,which accounted for more than 90% of the infected population.There is also a significant variation in HIV-1 subtype distribution between time periods,regions and populations in China.Highly active antiretroviral therapy can improve the quality of livingfor patients with HIV,but also to some extent accelerate the generation and spreading of resistant strains.Studies have showed that there is a certain correlation between subtypes and drug resistance,which has become a main subject research in AIDS in various countries.Chemokine CCR5 or/and CXCR4 are the auxiliary receptors that must be used when HIV infects the host target cells.Different genotypes of HIV-1 have different characteristics in the utilization of co-receptors,which has also become a hot issue in AIDS research in recent years.In this study,preliminary systematic and comprehensive understanding of the distribution characteristics between China HIV-1 subtypes and drug resistance in Jiangsu Province has been learned through Meta-analysis on HIV subtypes and drug resistance.Infectors in Southern Jiangsu,central and Northern Jiangsu were selected as the research object to carry out the investigation and study of molecular epidemiology in Jiangsu Province for a deep understanding of solution in HIV-1 subtype distribution and drug resistance and exploring the relationship between genotypes and drug resistance,combined with the relevant information of epidemiological investigation.What's more,HIV coreceptor prediction was used for predicting the utilization of HIV coreceptor,which has an important guiding significance for the prediction and prevention of AIDS epidemic in this province.Methods:1.Meta-analysis: A systematic literature review for studies of HIV-1 subtypes in English and Chinese through several databases were conducted in this Meta-analysis.Eligible articles were screened and selected based on the inclusion and exclusion criteria by two authors independently.Random-effects model was applied to calculate the pooled prevalence of different HIV-1 subtypes,and subgroup analysis examined prevalence estimates across subtypes,locations and populations.All meta-analyses were performed using Stata 19.0 software.2.HIV-1 subtype analysis:A total of 503 subjects,which have been confirmed to be HIV infections by enzyme-linked immunosorbent experiment and protein imprinting method?included people who have been treated and newly discovered infections?,were agreed to be recruited randomly in this study from Wuxi,Nantong and Yancheng in Jiangsu province between 2016-2017.EDTA blood samples were collected between 2016 and 2017.DNA was extracted from PBMC specimens and was amplified by nested PCR for the regions pol?env and gag.Samples of PCR positive were sent to companies for sequencing.Phylogenetic trees were constructed by using the adjacent connection method?Neighbor-Joining?and subtypes were determined finally by considering the subtypes of the three phylogenetic trees.3.Analysis of HIV-1 drug resistance?DR?mutations: 365 sequences of pol were submitted to HIV drug resistance database to infer drug-resistance mutations?DRMs?profile and antiretroviral susceptibility using Calibrated Population Resistance tool.?http://hivdb.stanford.edu?.4.Co-receptor prediction of HIV-1: Two types of online analysis tools were used to analyze the type of the crown tetrapeptide at V3 loop and to predict the usage of co-receptor among each subtype in Jiangsu province.5.Statistical analysis: All data were processed with SPSS 19.0 software.Using excel to establish database and SPSS 19.0 or SAS 9.3 software to analyze the data.Qualitative data were described with rate or proportion and analyzed with Chisquare test or fisher's exact test or Kruskal-Wallis test.Quantitative data were described with means and standard.The influence factors of HIV disease progression and drug-resistance were analyzed with repeated measurement data analysis,multivariate Logistic regression analyses or survival analyses.Differences were considered significant when P<0.05.Results:1.54 articles were selected and the pooled rate of drug resistance was 16.3% and the rate of primary drug resistance was 9.0%,the subtypes associated drug resistance were CRF01_AE,CRF07_BC,CRF08_BC,B/B' and C.The pooled rates of drug resistance of each subtype were13.4%,8.2%,15.0%,26.6%,33.3% and the rates of primary drug resistance of each subtype were 7.8%,5.3%,11.5%,15.6%,25.1%,respectively.Subgroup analysis showed that all subgroups showed a significant difference among groups?P<0.05?.The rates of primary resistance showed different characteristics among different subtypes,of which CRF01_AE/C showed the highest rates in northern China while CRF_BC/B/B' showed the highest in southwestern China.2.A total of 453 cases of the 503 HIV/AIDS patients were successfully genotyped,included CRF01_AE?45.5%,206/453??CRF07_BC?19.9%,90/453??B?6.4%,29/453??CRF67₀1B?7.7%,35/453??CRF08_BC?4.4%,20/453??CRF68₀1B?3.1%,14/453??CRF_BC?1.3%,6/453??C?0.7%,3/453??CRF02_AG?0.4%,2/453?.CRF01_AE?CRF07_BC?CRF67₀1B and B accounted for the lagest proportion of 79.7%.There was a significant variation in HIV-1 subtype distribution between sexes,ages,marriage and heterosexual/homosexual transmission?P<0.05?.3.122 samples had mutaitons associated resistance to drugs among 503 cases and the pool resistance rate was 24.3%?122/503?,among which transmitted drug resistance rate and primary drug resistance rate were 7.2%?6/83??31.3%?116/373?,respectively.K219E/N/Q/R werethe highest frequency of mutations in NRTIs,M184V/I?13.8%,16/116?accounted for the second;In NNRTIs,V179D?20.7%,24/116?and M230I/L?13.8%,16/116?were the main resistance mutation sites;In PR,M46I/L?13.8%,16/116?was the main resistance mutation site amongmajor drug resistance mutations while L24I?11.2%,13/116?was the main resistance mutation site amongsecondary drug resistance mutations.K65 R,Y115F,M184I/V,M230 L,Y181I/L,L100 I and L90 M could cause intermediate level /high level drug resistance in all kinds of drugs.4.The results showed that different subtypes of HIV infection distribution and drug resistance in the treatment group: several subtypes,CRF01_AE,CRF07_BC and CRF67₁B subtype of the resistant mutants had significant differences?P<0.05?,CRF08_BC and B subtypes in different resistance loci distribution without statistical significance?P>0.05?;Difference in distribution of subtypes in different degree of resistance were not statistically significant?P>0.05?.5.Crown tetrapeptide of v3 loop existed in 12 different types among 340 cases with env sequence.Analysis of V3 loop amino acid sequence revealed that CRF01_AE had the most complex crown tetrapeptide,included GPGQ,GPGR,GPGH,GPGK,GPGA and some other crown tetrapeptides,of which GPGQ accounted for the most?75.6%,121/160?;three crown tetrapeptides existed inB subtypes,included GPGR,GPGQ and GPGK,GPGR?65.0%,13/20?had the most proportion.CRF07_BC had GPGQ?97.0%?,GPGL?1.5%?,GSGQ?1.5%?.CRF08_BC had three kinds of crown tetrapeptides,as GPGQ?83.3%?,GPGR?11.1%?,GPRQ?5.6%?.6.In the 340 cases,45.6%?155/340?strains using CXCR4 as coreceptor.Among different subtypes,67.6%?108/160?of the CRF01_AE were predicted to be X4 and all CRF68₀1B were X4;Only 10%?2/20?of the B subtype using X4 as a coreceptor;The main strains of C and CRF07_BC subtypes were CCR5 cell tropism.7.CRF68₀1B and CRF01_AE infectors exsisted in lower CD4+ T cell counts,with 251cell/?l and 286cells/?l,respectively;infectors with B subtype showed the highest CD4+ T cell counts?427cells/?l?;Besides,The count level of CD4+ T cells in X4 cell tropism infected people was lower than that of CD4+ T cells in those infected with R5 cell tropism.Conclusion:1.The distribution of HIV genotypes in China wascomplexand theprevalenceof primary drug resistance of eachsubtypewas high,together with asignificantdifference amongsubtypes.It isnecessarytostrengthenthemonitoring of different subtypes of drug resistant strains in China to prevent the recombination andspreading ofresistant strains.2.In the past 2016-2017 years,there are many kinds of subtype epidemic strains in Jiangsu province.CRF01_AE,CRF 07_BC,CRF67₀1B and B strains are the dominant epidemic strains,and a variety of new recombinants are also popular in the region.Therefore,in order to prevent the further restructuring of various subtypes and the spread of drug-resistant strains,further health education and behavior intervention for HIV infected persons and AIDS population in Jiangsu Province should be strengthened,and continue to pay close attention to the dynamic distribution of HIV-1 subtypes in this population,especially in high-risk groups.3.Although the primary drug resistance of both NNRTIs and PIs were within the drug resistance alert line,the primary resistance rate of HIV-1 newly infected persons in this area was in a moderate epidemicon the overall level and there was more serious situation than ever.So,more attention should be taken to strengthen the new HIV infection of primary drug resistance detection,moreover,it suggested that resistant mutations detection should be implemented before receiving antiretroviral treatment.4.Drug resistance in treated population of HIV showed high levels,but mostly occured in patients with antiviral treatment for more than 2 years,indicated that antiviral treatment scheme in Jiangsu province was feasible.And strengthen the monitoring of antimicrobial resistance of the population was still a impotant thing,which will be benefit for adjusting the program in a timely manner to prevent drug resistance,cross resistance and the spreading of drug-resistant strains.5.The top four peptides of V3 loop type changed over 2016-2017 and there were many types of crown tetrapeptide of v3 loop.In each subype,GPGQ was still the main type of all except B subtype.In addition,The V3 gene in the CRF01_AE subtype had undergone some new variation.6.Different HIV subtypes showed different characteristics in the utilization of co-receptor.CRF01_AE subtype had a higher proportion of CXCR4 cell tropism and a lower level of CD4+ T cell count.X4 cell tropism infected people tended to have low level CD4+ T cell count.